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1.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445156

RESUMO

Cellular responses to micro- and hypergravity are rapid and complex and appear within the first few seconds of exposure. Transcriptomic analyses are a valuable tool to analyze these genome-wide cellular alterations. For a better understanding of the cellular dynamics upon altered gravity exposure, it is important to compare different time points. However, since most of the experiments are designed as endpoint measurements, the combination of cross-experiment meta-studies is inevitable. Microarray and RNA-Seq analyses are two of the main methods to study transcriptomics. In the field of altered gravity research, both methods are frequently used. However, the generation of these data sets is difficult and time-consuming and therefore the number of available data sets in this research field is limited. In this study, we investigated the comparability of microarray and RNA-Seq data and applied the results to a comparison of the transcriptomics dynamics between the hypergravity conditions during two real flight platforms and a centrifuge experiment to identify temporal adaptation processes. We performed a comparative study on an Affymetrix HTA2.0 microarray and a paired-end RNA-Seq data set originating from the same Jurkat T cell RNA samples from a short-term hypergravity experiment. The overall agreeability was high, with better sensitivity of the RNA-Seq analysis. The microarray data set showed weaknesses on the level of single upregulated genes, likely due to its normalization approach. On an aggregated level of biotypes, chromosomal distribution, and gene sets, both technologies performed equally well. The microarray showed better performance on the detection of altered gravity-related splicing events. We found that all initially altered transcripts fully adapted after 15 min to hypergravity and concluded that the altered gene expression response to hypergravity is transient and fully reversible. Based on the combined multiple-platform meta-analysis, we could demonstrate rapid transcriptional adaptation to hypergravity, the differential expression of the ATPase subunits ATP6V1A and ATP6V1D, and the cluster of differentiation (CD) molecules CD1E, CD2AP, CD46, CD47, CD53, CD69, CD96, CD164, and CD226 in hypergravity. We could experimentally demonstrate that it is possible to develop methodological evidence for the meta-analysis of individual data.


Assuntos
Hipergravidade , Linfócitos T/metabolismo , Transcriptoma , Humanos , Células Jurkat , RNA-Seq , Voo Espacial , Ativação Transcricional
2.
Sci Rep ; 11(1): 15927, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354210

RESUMO

Previous studies focusing on the age disparity in COVID-19 severity have suggested that younger individuals mount a more robust innate immune response in the nasal mucosa after infection with SARS-CoV-2. However, it is unclear if this reflects increased immune activation or increased immune residence in the nasal mucosa. We hypothesized that immune residency in the nasal mucosa of healthy individuals may differ across the age range. We applied single-cell RNA-sequencing and measured the cellular composition and transcriptional profile of the nasal mucosa in 35 SARS-CoV-2 negative children and adults, ranging in age from 4 months to 65 years. We analyzed in total of ~ 30,000 immune and epithelial cells and found that age and immune cell proportion in the nasal mucosa are inversely correlated, with little evidence for structural changes in the transcriptional state of a given cell type across the age range. Orthogonal validation by epigenome sequencing indicate that it is especially cells of the innate immune system that underlie the age-association. Additionally, we characterize the predominate immune cell type in the nasal mucosa: a resident T cell like population with potent antiviral properties. These results demonstrate fundamental changes in the immune cell makeup of the uninfected nasal mucosa over the lifespan. The resource we generate here is an asset for future studies focusing on respiratory infection and immunization strategies.


Assuntos
COVID-19/imunologia , Mucosa Nasal/imunologia , SARS-CoV-2/imunologia , Adolescente , Adulto , COVID-19/genética , Criança , Pré-Escolar , Feminino , Humanos , Imunidade Celular , Imunidade Inata , Lactente , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/citologia , Mucosa Nasal/metabolismo , Índice de Gravidade de Doença , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transcriptoma , Adulto Jovem
3.
Blood Cancer J ; 11(8): 142, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376633

RESUMO

This study evaluated the safety and immunogenicity of BNT162b2 vaccine in patients with hematological malignancies. Antibodies blocking spike binding to immobilized ACE-2 (NAb) correlated with anti-Spike (S) IgG d42 titers (Spearman r = 0.865, p < 0.0001), and an anti-S IgG d42 level ≥3100 UA/mL was predictive of NAb ≥ 30%, the positivity cutoff for NAb (p < 0.0001). Only 47% of the patients achieved an anti-S IgG d42 level ≥3100 UA/mL after the two BNT162b2 inocula, compared to 87% of healthy controls. In multivariable analysis, male patients, use of B-cell targeting treatment within the last 12 months prior to vaccination, and CD19+ B-cell level <120/uL, were associated with a significantly decreased probability of achieving a protective anti-S IgG level after the second BNT162b2 inoculum. Finally, using the IFN-γ ELISPOT assay, we found a significant increase in T-cell response against the S protein, with 53% of patients having an anti-S IgG-positive ELISPOT after the second BNT162b2 inoculum. There was a correlation between the anti-S ELISPOT response and IgG d42 level (Spearman r = 0.3026, p = 0.012). These findings suggest that vaccination with two BNT162b2 inocula translates into a significant increase in humoral and cellular response in patients with hematological malignancies, but only around half of the patients can likely achieve effective immune protection against COVID-19.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/complicações , COVID-19/imunologia , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/imunologia , Imunogenicidade da Vacina , SARS-CoV-2/imunologia , Imunidade Adaptativa , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/administração & dosagem , Comorbidade , Feminino , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto Jovem
4.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361000

RESUMO

The recruitment of T cells is a crucial component in the inflammatory cascade of the body. The process involves the transport of T cells through the vascular system and their stable arrest to vessel walls at the site of inflammation, followed by extravasation and subsequent infiltration into tissue. Here, we describe an assay to study 3D T cell dynamics under flow in real time using a high-throughput, artificial membrane-free microfluidic platform that allows unimpeded extravasation of T cells. We show that primary human T cells adhere to endothelial vessel walls upon perfusion of microvessels and can be stimulated to undergo transendothelial migration (TEM) by TNFα-mediated vascular inflammation and the presence of CXCL12 gradients or ECM-embedded melanoma cells. Notably, migratory behavior was found to differ depending on T cell activation states. The assay is unique in its comprehensiveness for modelling T cell trafficking, arrest, extravasation and migration, all in one system, combined with its throughput, quality of imaging and ease of use. We envision routine use of this assay to study immunological processes and expect it to spur research in the fields of immunological disorders, immuno-oncology and the development of novel immunotherapeutics.


Assuntos
Microfluídica/métodos , Linfócitos T/fisiologia , Migração Transendotelial e Transepitelial , Adesão Celular , Linhagem Celular Tumoral , Células Cultivadas , Quimiocina CXCL12/metabolismo , Endotélio Vascular/fisiologia , Matriz Extracelular/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
5.
Nutrients ; 13(7)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34371826

RESUMO

BACKGROUND: Sarcopenia is a major health problem in older adults. Exercise and nutrient supplementation have been shown to be effective interventions but there are limited studies to investigate their effects on the management of sarcopenia and its possible underlying mechanisms. Here, we studied T cell gene expression responses to interventions in sarcopenia. METHODS: The results of this study were part of a completed trial examining the effectiveness of a 12-week intervention with exercise and nutrition supplementation in community-dwelling Chinese older adults with sarcopenia, based on the available blood samples at baseline and 12 weeks from 46 randomized participants from three study groups, namely: exercise program alone (n = 11), combined-exercise program and nutrition supplement (n = 23), and waitlist control group (n = 12). T cell gene expression was evaluated, with emphasis on inflammation-related genes. Real-time PCR (RT-PCR) was performed on CD3 T cells in 38 selected genes. Correlation analysis was performed to relate the results of gene expression analysis with lower limb muscle strength performance, measured using leg extension tests. RESULTS: Our results showed a significant improvement in leg extension for both the exercise program alone and the combined groups (p < 0.001). Nine genes showed significant pre- and post-difference in gene expression over 12 weeks of intervention in the combined group. Seven genes (RASGRP1, BIN1, LEF1, ANXA6, IL-7R, LRRN3, and PRKCQ) showed an interaction effect between intervention and gene expression levels on leg extension in the confirmatory analysis, with confounder variables controlled and FDR correction. CONCLUSIONS: Our findings showed that T cell-specific inflammatory gene expression was changed significantly after 12 weeks of intervention with combined exercise and HMB supplementation in sarcopenia, and that this was associated with lower limb muscle strength performance.


Assuntos
Suplementos Nutricionais , Exercício Físico/fisiologia , Expressão Gênica/genética , Sarcopenia/terapia , Linfócitos T/metabolismo , Valeratos/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Análise Fatorial , Feminino , Humanos , Vida Independente , Extremidade Inferior/fisiopatologia , Masculino , Força Muscular/genética , Músculo Esquelético/fisiopatologia , Treinamento de Força/métodos , Sarcopenia/genética , Resultado do Tratamento
6.
Nat Methods ; 18(8): 965-974, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34341582

RESUMO

CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.


Assuntos
Sistemas CRISPR-Cas , Epigênese Genética , Epigenoma , Edição de Genes/métodos , Regulação da Expressão Gênica , Animais , Encéfalo/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Linfócitos T/metabolismo
7.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360833

RESUMO

CD39 is an enzyme which is responsible, together with CD73, for a cascade converting adenosine triphosphate into adenosine diphosphate and cyclic adenosine monophosphate, ultimately leading to the release of an immunosuppressive form of adenosine in the tumor microenvironment. Here, we first review the environmental and genetic factors shaping CD39 expression. Second, we report CD39 functions in the T cell compartment, highlighting its role in regulatory T cells, conventional CD4+ T cells and CD8+ T cells. Finally, we compile a list of studies, from preclinical models to clinical trials, which have made essential contributions to the discovery of novel combinatorial approaches in the treatment of cancer.


Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Linfócitos T/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/fisiologia , Apirase/genética , Apirase/imunologia , Apirase/fisiologia , Regulação da Expressão Gênica , Humanos , Linfócitos T/imunologia
8.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445309

RESUMO

In our previous work, we built the model of PPARγ dependent pathways involved in the development of the psoriatic lesions. Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor and transcription factor which regulates the expression of many proinflammatory genes. We tested the hypothesis that low levels of PPARγ expression promote the development of psoriatic lesions triggering the IL17-related signaling cascade. Skin samples of normally looking and lesional skin donated by psoriasis patients and psoriatic CD3+ Tcells samples (n = 23) and samples of healthy CD3+ T cells donated by volunteers (n = 10) were analyzed by real-time PCR, ELISA and immunohistochemistry analysis. We found that the expression of PPARγ is downregulated in human psoriatic skin and laser treatment restores the expression. The expression of IL17, STAT3, FOXP3, and RORC in psoriatic skin before and after laser treatment were correlated with PPARγ expression according to the reconstructed model of PPARγ pathway in psoriasis.In conclusion, we report that PPARγ weakens the expression of genes that contribute in the development of psoriatic lesion. Our data show that transcriptional regulation of PPARγ expression by FOSL1 and by STAT3/FOSL1 feedback loop may be central in the psoriatic skin and T-cells.


Assuntos
PPAR gama/metabolismo , Psoríase/metabolismo , Transdução de Sinais , Adulto , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interleucina-17/metabolismo , Masculino , Pessoa de Meia-Idade , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , PPAR gama/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T/metabolismo
9.
Biomolecules ; 11(8)2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34439887

RESUMO

The SARS-CoV-2 pandemic has completely disrupted the health systems of the entire planet. From the earliest months, it became increasingly clear that in addition to affecting the upper airways and lungs, there were other organs that could be affected. Among these, the skin became a real "sentinel signal" to be able to even suspect COVID-19. Background: this study deals with a little-explored issue for now: the study of skin immunopathology in SARS-CoV-2 positive subjects ascertained using the most reliable methods available. Methods: we used skin biopsy samples from SARS-CoV-2 positive and negative patients, studying morphology (Hematoxylin-Eosin), T lymphocyte population (CD4 and CD8), three markers such as HMGB-1, TIM-3 and HO-1 by immunohistochemistry. Results: although the presence of the CD4 and CD8 T population did not differ statistically significantly, we found greater activation and release of HMGB-1 in skin samples from SARS-CoV-2 positive patients, greater immunolabeling for TIM-3 at the level of CD4 and CD8 and a reduced expression of Heme oxygenase 1. Conclusions: these results support the possibility that there is immune deregulation in SARS-CoV-2 positive patients who develop skin manifestations of various kinds.


Assuntos
COVID-19/complicações , Proteína HMGB1/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Dermatopatias/metabolismo , Pele/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pele/patologia , Dermatopatias/etiologia , Dermatopatias/patologia , Linfócitos T/metabolismo
10.
Nat Commun ; 12(1): 5031, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413312

RESUMO

The limited sensitivity of Förster Resonance Energy Transfer (FRET) biosensors hinders their broader applications. Here, we develop an approach integrating high-throughput FRET sorting and next-generation sequencing (FRET-Seq) to identify sensitive biosensors with varying substrate sequences from large-scale libraries directly in mammalian cells, utilizing the design of self-activating FRET (saFRET) biosensor. The resulting biosensors of Fyn and ZAP70 kinases exhibit enhanced performance and enable the dynamic imaging of T-cell activation mediated by T cell receptor (TCR) or chimeric antigen receptor (CAR), revealing a highly organized ZAP70 subcellular activity pattern upon TCR but not CAR engagement. The ZAP70 biosensor elucidates the role of immunoreceptor tyrosine-based activation motif (ITAM) in affecting ZAP70 activation to regulate CAR functions. A saFRET biosensor-based high-throughput drug screening (saFRET-HTDS) assay further enables the identification of an FDA-approved cancer drug, Sunitinib, that can be repurposed to inhibit ZAP70 activity and autoimmune-disease-related T-cell activation.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fosfotransferases/metabolismo , Células Cultivadas , Humanos , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo
11.
Cell Rep ; 36(8): 109570, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34390647

RESUMO

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.


Assuntos
COVID-19/prevenção & controle , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Anticorpos Antivirais/sangue , Ligante de CD40/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-2/metabolismo , Peptídeos/imunologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Vacinação , Vacinas Sintéticas/imunologia
12.
Nat Commun ; 12(1): 4906, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385456

RESUMO

Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 (CDH12, N-Cadherin 2), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a (ITGA1), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.


Assuntos
Caderinas/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Imunoterapia/métodos , Neoplasias da Bexiga Urinária/terapia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Cateninas/genética , Cateninas/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Estimativa de Kaplan-Meier , Terapia Neoadjuvante/métodos , Avaliação de Resultados em Cuidados de Saúde , Proteômica/métodos , RNA-Seq/métodos , Linfócitos T/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/cirurgia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/cirurgia
13.
Nat Commun ; 12(1): 4907, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389726

RESUMO

The intestinal mucosa constitutes an environment of closely regulated immune cells. Dendritic cells (DC) interact with the gut microbiome and antigens and are important in maintaining gut homeostasis. Here, we investigate DC transcriptome, phenotype and function in five anatomical locations of the gut lamina propria (LP) which constitute different antigenic environments. We show that DC from distinct gut LP compartments induce distinct T cell differentiation and cytokine secretion. We also find that PD-L1+ DC in the duodenal LP and XCR1+ DC in the colonic LP comprise distinct tolerogenic DC subsets that are crucial for gut homeostasis. Mice lacking PD-L1+ and XCR1+ DC have a proinflammatory gut milieu associated with an increase in Th1/Th17 cells and a decrease in Treg cells and have exacerbated disease in the models of 5-FU-induced mucositis and DSS-induced colitis. Our findings identify PD-L1+ and XCR1+ DC as region-specific physiologic regulators of intestinal homeostasis.


Assuntos
Antígeno B7-H1/imunologia , Células Dendríticas/imunologia , Homeostase/imunologia , Mucosa Intestinal/imunologia , Receptores de Quimiocinas/imunologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Colite/genética , Colite/imunologia , Colite/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/imunologia , Homeostase/genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transcriptoma/genética , Transcriptoma/imunologia
15.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199417

RESUMO

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer treatment. Despite this achievement, mAbs-based therapies are struggling with limitations including poor tissue and tumor penetration, long half-life time, poor oral bioavailability, and expensive production costs, which prompted a shift towards the development of the small-molecule inhibitors of PD-1/PD-L1 pathways. Even though many small-molecule inhibitors targeting PD-1/PD-L1 interaction have been reported, their development lags behind the corresponding mAb, partly due to the challenges of developing drug-like small molecules. Herein, we report the discovery of a series of novel inhibitors targeting PD-1/PD-L1 interaction via structural simplification strategy by using BMS-1058 as a starting point. Among them, compound A9 stands out as the most promising candidate with excellent PD-L1 inhibitory activity (IC50 = 0.93 nM, LE = 0.43) and high binding affinity to hPD-L1 (KD = 3.64 nM, LE = 0.40). Furthermore, A9 can significantly promote the production of IFN-γ in a dose-dependent manner by rescuing PD-L1 mediated T-cell inhibition in Hep3B/OS-8/hPD-L1 and CD3-positive T cells co-culture assay. Taken together, these results suggest that A9 is a promising inhibitor of PD-1/PD-L1 interaction and is worthy for further study.


Assuntos
Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/citologia , Antígeno B7-H1/química , Linhagem Celular , Cristalografia por Raios X , Humanos , Interferon gama/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Cultura Primária de Células , Receptor de Morte Celular Programada 1/química , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
16.
Nat Commun ; 12(1): 4515, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312385

RESUMO

The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for 'reverse phenotyping'. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.


Assuntos
COVID-19/imunologia , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Linfócitos T/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , COVID-19/epidemiologia , COVID-19/virologia , Células Cultivadas , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , SARS-CoV-2/fisiologia , Linfócitos T/virologia
17.
Methods Mol Biol ; 2312: 3-14, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228281

RESUMO

CAR-T cell therapy is one of the most successful cell-based therapies. T cells are the most common cells to be genetically modified for cancer therapy, not only because T cells have cytotoxicity but also because they are easily cultured ex vivo and genetically modified with viral vectors. Hence, for nonexperts, T cell engineering is an ideal starting point for mammalian cell engineering or for development of therapeutics. Here, we have described a basic procedure for lentiviral transduction of human primary T cells to generate a CAR-T cell and assays to confirm CAR expression and function.


Assuntos
Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/genética , Linfócitos T/transplante , Transdução Genética , Separação Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Ativação Linfocitária , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
18.
Nature ; 595(7866): 289-294, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34194041

RESUMO

The global decline in malaria has stalled1, emphasizing the need for vaccines that induce durable sterilizing immunity. Here we optimized regimens for chemoprophylaxis vaccination (CVac), for which aseptic, purified, cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ) were inoculated under prophylactic cover with pyrimethamine (PYR) (Sanaria PfSPZ-CVac(PYR)) or chloroquine (CQ) (PfSPZ-CVac(CQ))-which kill liver-stage and blood-stage parasites, respectively-and we assessed vaccine efficacy against homologous (that is, the same strain as the vaccine) and heterologous (a different strain) controlled human malaria infection (CHMI) three months after immunization ( https://clinicaltrials.gov/ , NCT02511054 and NCT03083847). We report that a fourfold increase in the dose of PfSPZ-CVac(PYR) from 5.12 × 104 to 2 × 105 PfSPZs transformed a minimal vaccine efficacy (low dose, two out of nine (22.2%) participants protected against homologous CHMI), to a high-level vaccine efficacy with seven out of eight (87.5%) individuals protected against homologous and seven out of nine (77.8%) protected against heterologous CHMI. Increased protection was associated with Vδ2 γδ T cell and antibody responses. At the higher dose, PfSPZ-CVac(CQ) protected six out of six (100%) participants against heterologous CHMI three months after immunization. All homologous (four out of four) and heterologous (eight out of eight) infectivity control participants showed parasitaemia. PfSPZ-CVac(CQ) and PfSPZ-CVac(PYR) induced a durable, sterile vaccine efficacy against a heterologous South American strain of P. falciparum, which has a genome and predicted CD8 T cell immunome that differs more strongly from the African vaccine strain than other analysed African P. falciparum strains.


Assuntos
Anticorpos Neutralizantes/imunologia , Fígado/imunologia , Fígado/parasitologia , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/imunologia , Vacinas Atenuadas/imunologia , Adulto , Animais , Formação de Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Estágios do Ciclo de Vida/imunologia , Malária/sangue , Malária/imunologia , Malária/parasitologia , Malária/prevenção & controle , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/efeitos adversos , Vacinas Antimaláricas/química , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/crescimento & desenvolvimento , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Vacinação/efeitos adversos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/química
19.
Stem Cell Res Ther ; 12(1): 428, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321099

RESUMO

To date, two chimeric antigen receptors (CAR)-T cell products from autologous T cells have been approved by The United States Food and Drug Administration (FDA). The case-by-case autologous T cell generation setting is largely considered as a pivotal restraining cause for its large-scale clinical use because of the costly and prolonged manufacturing procedure. Further, activated CAR-T cells mainly express immune checkpoint molecules, including CTLA4, PD1, LAG3, abrogating CAR-T anti-tumor activity. In addition, CAR-T cell therapy potently results in some toxicity, such as cytokine releases syndrome (CRS). Therefore, the development of the universal allogeneic T cells with higher anti-tumor effects is of paramount importance. Thus, genome-editing technologies, in particular, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 are currently being used to establish "off-the-shelf" CAR-T cells with robust resistance to immune cell-suppressive molecules. In fact, that simultaneous ablation of PD-1, T cell receptor alpha constant (TRAC or TCR), and also ß-2 microglobulin (B2M) by CRISPR-Cas9 technique can support the manufacture of universal CAR-T cells with robust resistance to PD-L1. . Indeed, the ablation of ß2M or TARC can severely hinder swift elimination of allogeneic T cells those express foreign HLA-I molecules, and thereby enables the generation of CAR-T cells from allogeneic healthy donors T cells with higher persistence in vivo. Herein, we will deliver a brief overview of the CAR-T cell application in the context of tumor immunotherapy. More importantly, we will discuss recent finding concerning the application of genome editing technologies for preparing universal CAR-T cells or cells that can effectively counter tumor escape, with a special focus on CRISPR-Cas9 technology.


Assuntos
Receptores de Antígenos Quiméricos , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Imunoterapia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismo
20.
Neuropeptides ; 89: 102159, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34293596

RESUMO

T cells of aged people, and of patients with either cancer or severe infections (including COVID-19), are often exhausted, senescent and dysfunctional, leading to increased susceptibilities, complications and mortality. Neurotransmitters and Neuropeptides bind their receptors in T cells, and induce multiple beneficial T cell functions. Yet, T cells of different people vary in the expression levels of Neurotransmitter and Neuropeptide receptors, and in the magnitude of the corresponding effects. Therefore, we performed an individual-based study on T cells of 3 healthy subjects, and 3 Hepatocellular Carcinoma (HCC) patients. HCC usually develops due to chronic inflammation. The inflamed liver induces reduction and inhibition of CD4+ T cells and Natural Killer (NK) cells. Immune-based therapies for HCC are urgently needed. We tested if selected Neurotransmitters and Neuropeptides decrease the key checkpoint protein PD-1 in human T cells, and increase proliferation and killing of HCC cells. First, we confirmed human T cells express all dopamine receptors (DRs), and glutamate receptors (GluRs): AMPA-GluR3, NMDA-R and mGluR. Second, we discovered that either Dopamine, Glutamate, GnRH-II, Neuropeptide Y and/or CGRP (10nM), as well as DR and GluR agonists, induced the following effects: 1. Decreased significantly both %PD-1+ T cells and PD-1 expression level per cell (up to 60% decrease, within 1 h only); 2. Increased significantly the number of T cells that proliferated in the presence of HCC cells (up to 7 fold increase), 3. Increased significantly T cell killing of HCC cells (up to 2 fold increase). 4. Few non-conventional combinations of Neurotransmitters and Neuropeptides had surprising synergistic beneficial effects. We conclude that Dopamine, Glutamate, GnRH-II, Neuropeptide Y and CGRP, alone or in combinations, can decrease % PD-1+ T cells and PD-1 expression per cell, in T cells of both healthy subjects and HCC patients, and increase their proliferation in response to HCC cells and killing of HCC cells. Yet, testing T cells of many more cancer patients is absolutely needed. Based on these findings and previous ones, we designed a novel "Personalized Adoptive Neuro-Immunotherapy", calling for validation of safety and efficacy in clinical trials.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neuropeptídeos/farmacologia , Neurotransmissores/farmacologia , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/genética , Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/metabolismo , COVID-19/complicações , Carcinoma Hepatocelular/patologia , Dopamina/farmacologia , Agonistas de Dopamina/farmacologia , Humanos , Imunoterapia , Células Matadoras Naturais/metabolismo , Neoplasias Hepáticas/patologia , Receptores de Glutamato/efeitos dos fármacos , Receptores de Neuropeptídeos/metabolismo , Receptores de Neurotransmissores/metabolismo
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